Bone marrow stromal and anterior cruciate ligament remnant cell co‐culture‐derived extracellular vesicles promote cell activity in both cell types

Abstract The significance of anterior cruciate ligament (ACL) remnants during reconstruction remains unclear. Co‐culturing ACL remnant cells and bone marrow stromal cells (BMSCs) may reduce apoptosis and enhance hamstring tendon activity. This study investigated whether extracellular vesicles (EVs), which facilitate cell–cell interactions, act as the active components, improving graft maturation in this co‐culture. The effects of EVs on cell viability, proliferation, migration and gene expression in the rabbit ACL remnant cells and BMSCs were assessed using control (BMSC‐only culture), co‐culture (ACL remnant cells and BMSCs, CM) and co‐culture without EVs (CM ∆ EVs) media. EVs were isolated from control (BMSC‐EV) and co‐culture (CM‐EV) media and characterized. CM significantly enhanced the proliferation, migration and expression of transforming growth factor (TGF‐β)‐, vascular endothelial growth factor (VEGF)‐, collagen synthesis‐ and tenogenesis‐related genes. However, CM‐induced effects were reversed by the CM ∆ EVs treatment. CM‐EV treatment exhibited higher potential to enhance proliferation, migration and gene expression in the ACL remnant cells and BMSCs than BMSC‐EV and non‐EV treatments. In conclusion, EVs, secreted under the coexistence of ACL remnant cells and BMSCs, primarily increase the cell viability, proliferation, migration and gene expression of collagen synthesis‐, TGF‐β‐, VEGF‐ and tenogenesis‐related genes in both cell types.

treatment methods to restore mobility and knee function.However, ACL reconstruction may be required depending on the severity of the injury or the presence of other comorbidities, such as an intraarticular ligament or meniscus injury.The standard procedure for ACL reconstruction involves passing a tendon graft through a drilled tibia and femoral bone tunnel.The graft healing process comprises successive stages, including graft necrosis, vascularization, recellularization, remodelling and maturation. 4,5Nevertheless, despite advancements in surgical techniques, enhancing graft maturation and preventing graft retear following ACL reconstruction remain significant challenges.
Remnant preservation in ACL reconstruction improves graft maturation. 6,7However, whether ACL remnant preservation offers substantial benefits remains debatable within the medical community.For instance, Song et al. 8 highlighted the poor tissue structure and limited capability to enhance graft maturation with no biomechanical and biological advantages.0][11] In the intraarticular microenvironment, the ACL remnant is coated with bone marrow cells released from the bone tunnel, and the implanted graft is surrounded by the ACL remnant/ bone marrow mixture.Although the intricate relationship between ACL remnants and bone marrow cells in the intraarticular microenvironment plays a crucial role in graft maturation, the exact mechanism remains unknown.Lu et al. 12 first demonstrated that the ACL remnants upregulate the proliferation and gene expression associated with collagen synthesis and the tenogenesis of bone marrow stromal cells (BMSCs).Further, ACL remnant cell/BMSC co-culture medium could attenuate the apoptosis and increase the activity of the hamstring tendon and tenocyte. 13However, the effect of ACL remnant cell/BMSC co-culture medium on feedback to the ACL remnant cells and BMSCs is unknown, and the active component of the co-culture medium involved in graft maturation remains elusive.
Extracellular vesicles (EVs), which are secreted by cells to facilitate cell-cell interactions, contain abundant intercellular communication agents, such as mRNAs, microRNAs, lipids and proteins, which regulate the target cell activities. 14,15[18] Therefore, we hypothesized that the ACL remnant cell/BMSC co-culture medium, simulating the intraarticular microenvironment of ACL reconstruction, positively affect and upregulate ACL remnant cell and BMSC activities.We also investigated whether EVs, as the key active components, modulate the effects of the intraarticular microenvironment on effective graft maturation following ACL reconstruction.To test this hypothesis, in this study, we explored the role of EVs derived from ACL remnant cell/BMSC co-culture medium in ACL reconstruction on ACL remnant cells and BMSCs bioactivities.The present study reveals the intricate interactions between ACL remnant cells, BMSCs and EVs and provides critical insights into the understanding of graft maturation in ACL reconstruction procedures.

| Experimental design
In this study, we introduced the direct co-culture system of ACL remnant cells and BMSCs to simulate the intraarticular microenvironment of ACL reconstruction.The ACL remnant cells and BMSCs were harvested from six skeletally mature New Zealand male rabbits, as described previously. 13All animal experiments, tissue harvesting protocols and cell culturing were performed following the procedures described by Lu et al. 13 The third passage of ACL remnant cells (from the ACL-cut 4-week rabbit) and BMSCs was used in this study.All animal protocols were approved by the Institutional Animal Care and Use Committees (Kaohsiung Medical University; approval number KMU-107193).
Figure 1 illustrates the experimental design followed in this study.First, the cells were divided into three groups depending on their medium of growth-control medium (BMSC-only culture), coculture medium (ACL remnant cell and BMSC direct co-culture, CM) and co-culture medium without EVs (CM ∆ EVs)-to investigate the effects of ACL remnant cell and BMSC co-culture on proliferation, migration and expression of collagen I and III (COL-I & III), transforming growth factor (TGFβ), vascular endothelial growth factor (VEGF) and tenogenic genes (Scx, TNC).Next, the EVs were isolated from the control (BMSC-EV) and co-culture medium (CM-EV) and characterized based on size, morphology and expression of biological markers.
Finally, the effects of EVs on cell activity and gene expression were assessed in ACL remnant cells and BMSCs after BMSC-EV and CM-EV treatment compared with those of the non-EV treatment group.

| Cell proliferation and migration
The cell proliferation ratio of the ACL remnant cells and BMSCs in response to control medium (n = 6), CM (n = 6) and CM ∆ EVs (n = 6) were measured using a Click-iT EdU Cell Proliferation Kit (Cat.No. 1906238, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions. 12,13 investigate the change in cell migration rate following different media treatments to the ACL remnant cells and BMSCs (n = 6, each), transwell and scratch migration tests were employed.
The transwell assay was performed using a 6.5-mm transwell chamber with a pore size of 8 μm (Cat.No. 3422, Costar, Corning Inc., Corning, NY, USA). 12,13The migrated cells were fixed by 4% formaldehyde for 15 min, washed with PBS twice and added the methanol 10 min.Then, the migrated cells were stained with 0.1% crystal violet for 30 min.The migrated cells stained in purple were counted under a microscope.The relative migration rate was calculated as the ratio of the migration cells in each treatment group compared to that of the control group (% of the control).The scratch migration test was assessed by observing the closure of the scratch gap at regular intervals (12, 24, 36   and 48 h) under a microscope (Leica DMI6000B; Leica Microsystems, Germany). 12,13No. K1642, Thermo Fisher Scientific Waltham, MA, USA) following the manufacturer's instructions. 13The primers, cycling conditions and quantification methods were as described previously. 13The primers used for cDNA synthesis are presented in Table S1.

| Immunofluorescence staining of COL-I & III, TGFβ and VEGF
The ACL remnant cells and BMSCs treated with control medium (n = 6), CM (n = 6) and CM ∆ EVs (n = 6) were assessed for the The primary antibodies, fluorescent secondary antibodies, and the staining procedure were performed as described previously. 13,19The primary antibody used in this study are as follows: anti-COL-I ( 1

| Isolation and characterization of EVs from the BMSC-only culture and co-culture media
On Day 7 of culture, EVs were isolated from the BMSC-only culture and CM using a serial centrifugation method. 20,21Briefly, the culture medium (500 mL) was centrifuged at 20,000 × g for 30 min at 4°C to remove large cell fragments.Subsequently, the supernatant was collected and centrifuged at 120,000 × g for 90 min at 4°C to pellet the membrane-bound vesicles.The medium (supernatant) was removed, and the discarded medium without the membrane-bound vesicles represented the CM ∆ EVs.The pellet was washed with 10 mL of phosphate-buffered saline (PBS) and subjected to centrifugation at 120,000 × g for 90 min at 4°C.Then, the supernatant was discarded, and the pellet (EVs) was collected and re-suspended in 1 mL of PBS and filtered with a 0.22μm filter membrane.

| Characterization of EVs
The BMSC-EV and CM-EV were evaluated for size distribution, expression of biological markers, morphology and uptake by the ACL remnant cells and BMSCs.

| Size distribution
Particle size analysis was performed using a particle analyser (ZetaView® NTA-Nanoparticle Tracking Video Microscope PMX-130, Particle Metrix, Ammersee, Germany) following the manufacturer's protocol.were all obtained from Proteintech.The labelled proteins were visualized using a ChemiDoc™ XRS imaging system (Bio-Rad, Hercules, Cal, USA).

| Transmission electron microscopy analysis
EV morphology was observed using a transmission electron microscope (Hitachi®, Tokyo, Japan), and the images were captured using a digital camera (Olympus®, Tokyo, Japan).
Thereafter, EVs were rinsed with PBS, centrifuged at 120,000 × g for 90 min at 4°C, re-suspended in 1 mL serum-free medium and filtered using a 0.22μm filter membrane for further use.
EVs were added to the cells (10 10 EV particles/10 4 cells) for 6 h, and the treated cells were washed with PBS and fixed in 4% paraformaldehyde for 10 min.After subsequent washing with PBS twice, 100 μL of 1X phalloidin conjugate working solution (Cat No. ab176753, Abcam, Cambridge, MA, USA) was added to the cells to stain the cytoskeleton.The cell slides were mounted and stained with Hoechst 33342 (Sigma-Aldrich, St. Louis, MO, USA) and observed using a confocal microscope (Olympus IX-81-FV100, Olympus, Tokyo, Japan).

| Statistical analysis
All data are presented as the mean ± standard deviation (SD) with three measurements.In this study, the numerical value for the control group was set as 1, and the numerical values of the other experimental groups are presented in reference to the control group.
One-way analysis of variance with a subsequent Tukey post hoc test was used to determine the significant differences in multiple comparisons.All statistical analyses were performed using SPSS software version 20 (IBM, USA).Statistical significance was set at p < 0.05.

| Co-culture medium treatment significantly enhanced the proliferation, migration and gene expression both in ACL remnant cells and BMSCs
The cell proliferation (Edu expression), Ki67 gene expression and migration capability in both ACL remnant cells (Figure 2) and BMSCs (Figure 3) were significantly increased following CM treatment compared with those after BMSC-only culture medium treatment.The expression of COL-I & III-, TGFβ-, VEGF-and tenogenesis-related (Scx, TNC) genes was significantly improved after CM treatment.
However, the CM-induced increases in cell proliferation, migration and gene expression were significantly reversed by the CM ∆ EVs treatment (Figures 2 and 3).CM-EV revealed the potential of these cells to uptake and internalize the EVs (Figure 4B).

| Treatment with EVs improved the viability, proliferation, migration and gene expression in ACL remnant cells and BMSCs
As shown in Figures 5 and 6, ACL remnant cells and BMSCs treated with BMSC-EV and CM-EV showed higher cell viability, proliferation, migration and expression of COL-I & III, TGFβ, VEGF, Scx and TNC genes than those treated with non-EVs (control).These effects were more pronounced in ACL remnant cells treated with CM-EV than those in the cells treated with BMSC-EV, with significant differences in proliferation, migration and gene expression of COL-I, TGFβ and VEGF (Figure 5).Similarly, all these effects were more pronounced in BMSCs treated with CM-EV than those treated with BMSC-EV, with significant differences in cell viability, migration and gene expression of COL-I & III, TGFβ and TNC (Figure 6).

| DISCUSS ION
In this study, we investigated the effect of ACL remnant cell/BMSC co-culture medium, along with its main activator, which simulates the ACL remnant tissue covered with BMSCs during ACL reconstruction.Notably, CM significantly enhanced the proliferation, migration, collagen synthesis and tenogenesis capabilities of both the ACL remnant cells and BMSCs.Furthermore, the contrasting effects of CM ∆ EVs treatment confirmed EVs as the major active ingredient in the CM underlying the beneficial effects of the co-culture medium.The CM-EV uptake by the ACL remnant cells and BMSCs significantly enhanced cell viability, proliferation, migration and expression of collagen synthesis-, TGFβ-, VEGF-and tenogenesisrelated genes compared with BMSC-EV and non-EV uptakes.
In the intraarticular microenvironment, the ACL remnant tissue is coated with bone marrow from the drilled bone tunnel and the mixture surrounds the hamstring tendon graft during ACL reconstruction.Notably, the ACL remnants exhibit the paracrine potential of regulating the surrounding BMSCs toward increased collagen synthesis and tenogensis. 12Furthermore, Lu et al. 13 demonstrated that the ACL remnant cell/BMSC co-culture medium simultaneously attenuated the apoptosis and enhanced the activity of the hamstring tendon and tenocyte.In the present study, we demonstrated that the co-culture medium-treated ACL remnant cells and BMSCs presented increased cell activity and gene expression capability.In summary, the ACL remnant cell/BMSC co-culture medium promotes the activity of hamstring tendon and tenocytes and also provides feedback to enhance ACL remnant cells and BMSCs activities.Therefore, the mixture of ACL remnant and bone marrow cells may improve remnant preservation and promote implanted tendon graft maturation during ACL reconstruction.
The co-culture systems (indirect and direct) were introduced in the laboratory studies to observe the cell-cell interactions.A previous study, using the indirect co-culture system, demonstrated the paracrine effects and BMSC upregulation (lower chamber) by ACL remnant cells (upper chamber). 12However, the indirect co-culture system reflects only unidirectional signal transmission but not the F I G U R E 5 Cell viability, proliferation, migration and expression of collagen I and III-, TGFβ-, VEGF-and tenogenic-related genes in ACL remnant cells following treatment with EVs derived from BMSC-only culture medium (BMSC-EV), ACL remnant cell/BMSC co-culture medium (CM-EV) and non-EV medium (control) group.Migration is evaluated via transwell and scratch migration assays.In the transwell assay, cells that traverse the transwell membrane are stained in purple with crystal violet, and their migration ratio is determined.In the scratch assay, results are presented as the percentage of initial scratch closure.*p < 0.05; **p < 0.01.bidirectional response to the ACL remnant cells and BMSCs in the intraarticular microenvironment during ACL reconstruction.In a direct co-culture system, cell-cell interactions occur through direct gap junctions and autocrine, paracrine or endocrine signalling. 25,26reover, during cell-cell interaction, various secretomes present in the medium transfer different signals. 25,26Among these secretomes, EVs play an important role in cellular communication and signal transfer.In this study, we directly co-cultured the ACL remnant cells and BMSCs to mimic the clinical environment of ACL reconstruction.The ACL remnant cell/BMSC co-culture medium showed high capability to bidirectionally enhance the ACL remnant cells and BMSCs, which declined significantly after the removal of the EVs.
These results indicated that CM-EV facilitated the interactions between the ACL remnant cells and BMSCs in the direct co-culture system.Therefore, EVs were identified as the main activators within the co-culture medium.
EVs, containing variable mRNAs, microRNAs, lipids and proteins, derived from the stem cell culture medium exhibit a high potential to enhance injured tissue repair and cellular activity with a synergic effect. 14,15,27,289][30][31] Qi et al. 29 investigated a novel purified exosome product and found that the tenocytes could uptake these exosomes with increasing cell proliferation, tenogenic marker expression and collagen deposition.Furthermore, Shi et al., 30 applying the purified exosome product patch in a tendon-repair ex vivo model, demonstrated that it enhanced ultimate failed strength, reduced healing gap, increased COL-III gene expression and decreased inflammatory response.In the rat patella tendon defect study, EV treatment enhanced tendon healing by reducing inflammation and tenocyte apoptosis, increasing tenogenic progenitor cell proportion, enhancing the expression of collagen and tenogenic marker genes, and improving histological collagen alignment. 31In our study, we harvested EVs from the BMSC-only culture medium and ACL remnant cell/ BMSC co-culture medium.Treatment with these EVs increased the cell viability, proliferation, migration and gene expression of collagen synthesis, TGFβ, VEGF and tenogenic markers in ACL remnant cells and BMSCs.Moreover, CM-EV exhibited a higher potential to enhance the activities of ACL remnant cells and BMSCs compared with BMSC-EV.Nevertheless, EV components are not static, and their production varies depending on stimulation conditions, culture environments and cell interactions. 25,32 this study, we used the EV-free medium in all culture conditions to eliminate the interference of the EV-containing conventional medium.The EVs released following the crosstalk between ACL remnant cells and BMSCs exhibited higher potential for increasing cell activity within the microenvironment of ACL reconstruction compared with those released from the BMSC-only medium.Therefore, CM-EV may be used in ACL reconstruction to improve ACL remnant cells and BMSCs activities and consequently enhance graft maturation.
The present study has some limitations.First, we only investigated the effects of EVs on ACL remnant cells and BMSCs in vitro, and the enhancing capability of CM-EV should be validated using animal studies.Second, the components within the ACL remnant cell/BMSC co-culture medium were not evaluated.This study investigates EVs extracted from ACL cells/BMSCs co-culture and BMSCs F I G U R E 6 Cell viability, proliferation, migration and expression of collagen I and III-, TGFβ-, VEGF-and tenogenic-related genes in BMSCs following treatment with EV derived from BMSC-only culture medium (BMSC-EV), ACL remnant cell/BMSC co-culture medium (CM-EV) and non-EV medium (control) groups.Migration is evaluated via transwell and scratch migration assays.In the transwell assay, cells that traverse the transwell membrane are stained in purple with crystal violet, and their migration ratio is determined.In the scratch assay, results are presented as the percentage of initial scratch closure.*p < 0.05; **p < 0.01.4][35] EVs are composed of various components such as proteins, microRNAs and lipids, each extensively studied in the literature.However, there is no definitive conclusion regarding whether the effects of EVs arise from individual components or from their synergistic interactions. 36,37entifying specific EV components and their mechanisms of action would necessitate a separate, more comprehensive research effort, which represents a limitation of the current study.Third, we performed direct monolayer co-culture using ACL remnant cells and BMSCs.Consequently, further studies using a 3D co-culture system are essential to clarifying the relationship between ACL remnant cells, bone marrow cells and tendon grafts.
In conclusion, under the coexistence of ACL remnant cells and BMSCs, the secreted EVs increase cell viability, proliferation, migration and gene expression of COL-I & III, TGFβ and TNC in both cell types, which may be beneficial for the maturation of the implanted graft in ACL reconstruction.These findings hold high translational potential, offering insights and applications that could directly benefit patients with ACL reconstruction.The EVs derived from the co-culture medium could be a potential biomaterial to improve graft maturation following ACL reconstruction.

F I G U R E 1
Experimental design of the study.ACL, anterior cruciate ligament; BMSC, bone marrow stromal cell; CM, co-culture medium; EV, extracellular vesicle.immunofluorescence expression of COL-I & III, TGFβ and VEGF.

| 5 of 10 LIN
We investigated the effects of EVs derived from different culture media (BMSC-EV and CM-EV) on the viability (MTT assay, Cat.No. M2003, Sigma-Aldrich, St. Louis, MO, USA), proliferation (Edu assay, Ki67 gene expression), migration (transwell and scratch migration assays), gene expression (COL-I & III, TGFβ, VEGF, Scx, TNC) and immunofluorescence (collagen I and III, TGFβ, VEGF) of ACL remnant cells and BMSCs.The EV treatment concentration was maintained at 10 10 particles/10 4 cells for all experiments.The exosome-depleted FBS medium-treated cells were set as a control.et al.

| 7 of 10 LIN
EVs from BMSC-only culture medium and CM were isolated.EVs isolated from BMSC-only culture medium and CM exhibited an average particle size of 131.1 ± 111.6 and 128.3 ± 145.8 nm, respectively (Figure 4A, upper panel).After purification, the BMSC-EV and CM-EV expressed CD9, CD63, CD81, Alix and TSG101, in contrast to the BMSC and Co-culture media supernatant, which did not show α-tubulin expression(Figure 4A, lower panel [left]).Moreover, BMSC-EV and CM-EV were spherical in shape with central hypodense (light colour) and peripheral hyperdense (dark colour) regions (Figure4A, lower panel [right]), as determined using microscopic images.Collectively, these results indicated that EVs from both BMSC-only culture medium and CM were successfully isolated and that isolated EVs met the MISEV 2014 and 2018 criteria.[22][23][24]Furthermore, the treatment of ACL remnant cells and BMSCs with F I G U R E 2 Cell proliferation, migration and expression of collagen I and III-, TGFβ-, VEGF-and tenogenic-related genes in ACL remnant cells following treatment with BMSC-only culture medium (control), ACL remnant cell/BMSC co-culture medium (CM) and co-culture medium without extracellular vesicles (CM ∆ EVs).Migration is evaluated via transwell and scratch migration assays.In the transwell assay, cells that traverse the transwell membrane are stained in purple with crystal violet, and their migration ratio is determined.In the scratch assay, results are presented as the percentage of initial scratch closure.*p < 0.05; **p < 0.01.F I G U R E 3 Cell proliferation, migration and expression of collagen I and III-, TGFβ-, VEGF-and tenogenic-related genes in BMSCs following treatment with BMSC-only culture medium (control), ACL remnant cell/BMSC co-culture medium (CM) and co-culture medium without extracellular vesicles (CM ∆ EVs).Migration is evaluated via transwell and scratch migration assays.In the transwell assay, cells that traverse the transwell membrane are stained in purple with crystal violet, and their migration ratio is determined.In the scratch assay, results are presented as the percentage of initial scratch closure.*p < 0.05; **p < 0.01.F I G U R E 4 (A) Particle size distribution, expression levels of biomarkers and transmission electron microscopic images of EVs derived from BMSC-only culture (BMSC-EV) and ACL remnant cell/BMSC co-culture medium (CM-EV).(B) Immunofluorescence pictures show the CM-EV uptake and internalization by the ACL remnant cells and BMSCs.et al.